-
MMaarriinnee EEnnvviirroonnmmeenntt aanndd EEccoollooggyy
Molecular tools for detection of marine pests:
Musculista senhousia, Corbula gibba and Perna canaliculus
quantitative PCR assays.
Nathan J. Bott and Danièle Giblot-Ducray
SARDI Publication No. F2010/000991-1 SARDI Research Report
Series No. 522
SARDI Aquatic Sciences PO Box 120 Henley Beach SA 5022
February 2011
Report prepared for Biosecurity SA
-
ii
Molecular tools for detection of marine pests: Musculista
senhousia, Corbula gibba and Perna
canaliculus quantitative PCR assays.
Report prepared for Biosecurity SA
Nathan J. Bott and Danièle Giblot-Ducray
SARDI Publication No. F2010/000991-1 SARDI Research Report
Series No. 522
February 2011
-
This publication may be cited as: Bott, N. J. and Giblot-Ducray,
D (2011). Molecular tools for detection of marine pests: Musculista
senhousia, Corbula gibba and Perna canaliculus quantitative PCR
assays. Report prepared for Biosecurity SA. South Australian
Research and Development Institute (Aquatic Sciences), Adelaide.
SARDI Publication No. F2010/000991-1. SARDI Research Report Series
No. 522. 22pp. South Australian Research and Development Institute
SARDI Aquatic Sciences 2 Hamra Avenue West Beach SA 5064 Telephone:
(08) 8207 5400 Facsimile: (08) 8207 5406 http://www.sardi.gov.au
DISCLAIMER The authors warrant that they have taken all reasonable
care in producing this report. The report has been through the
SARDI Aquatic Sciences internal review process, and has been
formally approved for release by the Chief, Aquatic Sciences.
Although all reasonable efforts have been made to ensure quality,
SARDI Aquatic Sciences does not warrant that the information in
this report is free from errors or omissions. SARDI Aquatic
Sciences does not accept any liability for the contents of this
report or for any consequences arising from its use or any reliance
placed upon it. © 2011 SARDI This work is copyright. Apart from any
use as permitted under the Copyright Act 1968 (Cth), no part may be
reproduced by any process, electronic or otherwise, without the
specific written permission of the copyright owner. Neither may
information be stored electronically in any form whatsoever without
such permission. Printed in Adelaide: February 2011 SARDI
Publication No. F2010/000991-1 SARDI Research Report Series No. 522
Author(s): Nathan J. Bott and Danièle Giblot-Ducray Reviewer(s):
Marty Deveney and Kathryn Wiltshire Approved by: Jason Tanner
Principal Scientist – Marine Environment & Ecology Signed:
Date: 10 February 2011 Distribution: Biosecurity SA, SAASC Library
and University of Adelaide Library Circulation: Public Domain
iii
http://www.sardi.gov.au/
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Table of Contents Executive
Summary.............................................................................................................1
Acknowledgements
.............................................................................................................2
Introduction..........................................................................................................................3
Musculista senhousia
...................................................................................................4
Corbula gibba
...............................................................................................................4
Perna canaliculus
.........................................................................................................5
Molecular testing methods for marine pests
.................................................................6
Methods...............................................................................................................................8
Quantitative PCR (qPCR) assay design
.......................................................................8
Samples
.......................................................................................................................9
DNA extractions
...........................................................................................................9
Quantitative
PCR........................................................................................................10
Results
..............................................................................................................................10
Primers and
probes........................................................................................................10
Musculista senhousia
.................................................................................................12
Corbula gibba
.............................................................................................................14
Perna canaliculus
.......................................................................................................16
Discussion
.........................................................................................................................17
Conclusions and future approaches
...........................................................................18
References
........................................................................................................................20
Glossary of Terms
.............................................................................................................22
Figure 1: Asian date mussel, Musculista senhousia
............................................................4
Figure 2: European clam, Corbula gibba
.............................................................................5
Figure 3: New Zealand Greenlip mussel, Perna canaliculus
...............................................6 Figure 4: SARDI
Diagnostics laboratory
..............................................................................9
Figure 5: Amplification plot for Musculista senhousia qPCR assay
specificity testing. ......13 Figure 6: Amplification plot for
Corbula gibba qPCR assay specificity testing. ..................15
Figure 7: Amplification plot for Perna canaliculus qPCR assay
specificity testing .............17
Table 1: Primers and TaqMan MGB
probes....................................................................................
11 Table 2: Results of specificity testing for M. senhousia qPCR
assay.............................................. 13 Table 3:
Results of specificity testing for C. gibba qPCR
assay...................................................... 14
Table 4: Results of specificity testing for P. canaliculus qPCR
assay............................................. 16
iv
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Executive Summary The National System for the Prevention and
Management of Marine Pest Incursions requires tools
for the detection and monitoring of marine pests. Specific,
robust molecular assays for the
identification and quantification of marine pests (including
eggs and larval stages) from
environmental samples facilitate rapid, low-cost surveillance to
be undertaken and effective control
strategies to be implemented where marine pest incursions are
detected. The use of quantitative
Polymerase Chain Reaction (qPCR) techniques will be suitable for
this purpose. The polymerase
chain reaction (PCR) is an enzymatic technique used for the
amplification of nucleic acids (e.g.
DNA), and qPCR is a PCR technique monitored in real-time through
changes in fluorescence.
Currently, detection of marine pests is based primarily on
traditional survey techniques such as
observational walks, snorkel and dive surveys, plankton trawls,
traps and netting, with manual
sorting and identification. The use of qPCR offers the ability
to conduct testing of very large
numbers of samples to rapidly identify the genetic material of
the targeted organisms (referred to
as high-throughput screening). The successful development and
implementation of these methods
will allow for the testing of plankton samples to rapidly verify
the presence or absence of potential
pest species in marine waters. This study details the
development and assessment of qPCR
assays for the Asian Date Mussel, Musculista senhousia, the New
Zealand Greenlip Mussel, Perna
canaliculus and the European clam, Corbula gibba.
We have designed a qPCR assay that is, based on available
controls, specific to M. senhousia.
We have adapted a specific PCR assay for P. canaliculus to a
qPCR assay that is, based on
available controls, specific to P. canaliculus. We have further
tested the specificity of the C. gibba
qPCR assay, developed by Ophel-Keller et al. (2007), with
closely related corbulid species, and
based on available specimens this assay is specific to C.
gibba.
In the future, these qPCR assays, along with other marine pest
qPCR assays, are expected to be
used to monitor ports for marine pests, using DNA extracted from
plankton samples. Specific
diagnosis of marine pests is central to rapidly establishing the
distribution and prevalence of
marine pest species to manage and restrict their spread, and
monitoring changes in marine pest
distribution spatially and temporally facilitates targeted
eradication and control programmes where
feasible.
1
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Acknowledgements Assistance from the following individuals is
greatly acknowledged:
• Dr Paul Fisher and Bradley Pease (Queensland Department of
Employment, Economic
Development and Innovation) for the provision of mytilid bivalve
samples.
• Dr Richard Willan (NT Museum) for identifiying Musculista
senhousia samples and Dr
Jawahar Patil (CSIRO Marine Research) for providing M. senhousia
DNA.
• Mr Anders Hallan (University of Wollongong) for providing
corbulid specimens.
• Dr Alan McKay, Dr Kathy Ophel-Keller, Dr Herdina and Ms Teresa
Mammone (SARDI
Diagnostics) for assistance and advice in the laboratory.
• Dr Maylene Loo (SARDI Aquatic Sciences) for assistance in
obtaining M. senhousia
samples from Singapore.
• Dr Marty Deveney and Ms Kathryn Wiltshire (SARDI Aquatic
Sciences)
• Dr Michael Sierp, Mr John Gilliland and Mr Vic Neverauskas
(Biosecurity SA).
2
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Introduction The National System for the Prevention and
Management of Marine Pest Incursions requires tools
for the detection and monitoring of marine pests. Specific,
robust molecular assays for the
identification and quantification of marine pests (including
eggs and larval stages) from
environmental samples facilitate rapid, low-cost surveillance,
and inform effective control strategies
where marine pest incursions are detected.
Marine pests have the potential to cause significant harm to
endemic biodiversity and habitats
(Galil, 2007; Wallentinus and Nyberg, 2007). Marine pests can be
translocated and introduced by
numerous vectors including ship ballast, hull fouling, floating
debris and man-made structures such
as drilling platforms and canals (Bax et al., 2003). Marine pest
introductions continue to occur and
threaten the marine environment and associated industries (Hayes
and Sliwa, 2003). With
increasing globalisation comes faster and more frequent shipping
and air transport of live seafood.
Propagule pressure is only likely to increase unless effective
strategies are employed for early
detection, prevention and control. Central to such strategies is
the ability to rapidly identify the
presence of a particular pest species.
The development and implementation of rapid, sensitive and
accurate diagnostic techniques for
the identification and surveillance of marine pests from
environmental samples (e.g. sea water,
sediments, and ship ballast), is an essential step in early
detection and control of marine pests, to
maintain the status of pest-free areas and to limit the economic
impacts of management in areas
where pest are established and cannot be eradicated..
Current marine pest diagnostics research at SARDI includes the
development and refinement of
specific, sensitive, quantitative Polymerase Chain Reaction
(qPCR) assays for the detection of
several marine pest species. PCR is an enzymatic technique used
for the amplification of nucleic
acids (e.g. DNA) and qPCR is a PCR technique monitored in
real-time through changes in
fluorescence. Through consultation between SARDI Aquatic
Sciences and Biosecurity SA it was
decided to undertake a study to implement and/or develop qPCR
assays for three marine pest
species of significance to Australia, two of which: European
clam, Corbula gibba ; and Asian Bag
Mussel, Musculista senhousia, are part of the Consultative
Committee on Introduced Marine Pest Emergencies (CCIMPE) Trigger
List, which is endorsed by the National Introduced Marine Pest
Coordinating Group (NIMPCG). The third is the New Zealand
Greenlip Mussel, Perna canaliculus,
which has high potential as an invasive species and has been
previously reported but eradicated
from Outer Harbor and a hopper barge docked in Port Adelaide
(Wiltshire et al., 2010).
3
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Musculista senhousia Musculista senhousia (Figure 1) is commonly
known as the Asian date mussel and is native to the Pacific Ocean,
inhabiting coastal areas from Siberia and the Kuril Islands south
to Singapore
(Slack-Smith and Brearley, 1987). Aggregations can attain very
high densities in intertidal and
subtidal soft sediments, typically 5 000-10 000/m2, and up to 15
000/m2 (Crooks and Soule, 1999;
Dexter and Crooks, 2000; Reush and Williams, 1998). When in high
densities, M. senhousia can
cause significant habitat alteration, which can have profound
effects on native infaunal
communities, and native seagrasses (Crooks, 1998). Based on
seawater temperature, M.
senhousia has significant potential to become established across
Australian waters
(http://marinepests.gov.au/__data/assets/pdf_file/0005/952529/Musculista-ncp-08.pdf).
Figure 1: Asian date mussel, Musculista senhousia
Corbula gibba The European clam, C. gibba (see Figure 2), is
commonly found in subtidal environments, in
coastal and estuarine silts and muddy gravels, in its native
Europe (Holmes and Miller, 2006).
Very high densities of C. gibba have been recorded in Port
Phillip Bay (up to 2600/m2), and have
been linked to changes in benthic community structure (Currie
and Parry, 1999). In Europe, high
abundances of C. gibba tend to be associated with habitat
degradation, although this has not been
adequately studied in Australia.
4
-
Figure 2: European clam, Corbula gibba
Perna canaliculus Perna canaliculus (Figure 3) is commonly known
as the New Zealand greenlip mussel and is
endemic to New Zealand waters, it has been previously reported
from Outer Harbor and from a
vessel in Port Adelaide, but it was successfully eradicated
(Wiltshire et al., 2010). Perna
canaliculus is commercially harvested as seafood in New Zealand.
Potential impacts of P.
canaliculus on native populations are unknown but it would
likely include competition with native
bivalve species, fouling structures and potential introduction
of diseases to the Australian mollusc
aquaculture industry.
5
-
Figure 3: New Zealand Greenlip mussel, Perna canaliculus
Molecular testing methods for marine pests Development of rapid
testing methods for marine pests has recently focussed on
molecular
techniques. A broad range of these techniques have been
developed for marine pests (see Bott et
al. 2010 and references therein). Polymerase Chain Reaction
(PCR) has revolutionised many
areas of biological research including species and strain
delineation. PCR can amplify minute
amounts of template DNA, and its high specificity makes it
highly effective for species and strain
identification for a wide range of organisms. The relatively low
cost of equipment and reagents
makes PCR accessible to a wide range of laboratories.
Quantitative PCR (qPCR) allows the
amplification of a target DNA to be monitored in real-time as
amplification occurs. qPCR offers a
relatively rapid analysis (< 2 hours), the potential for
high-throughput, allows linear quantification
over a wide dynamic range (>6 orders of magnitude), and has
the benefit of not requiring post-
PCR handling (“closed-tube” format), decreasing the likelihood
of sample contamination. It is now
routinely used in numerous clinical applications for the
detection of a wide range of bacterial,
fungal, parasitic and viral diseases of humans (Espy et al.,
2006). Recent advances have seen a
6
-
number of studies utilising qPCR-based techniques for the
identification of marine pests (see
Galluzi et al., 2004; Pan et al., 2008).
The development of these tests requires that the target organism
is taxonomically unambiguous.
Testing of a number of species closely related to the target
organism is required as well as testing
of environmental samples containing unknown taxa. Most test
development achieves the first
criterion but for implementation, it is important to validate
tests on samples exhibiting higher
complexity such as natural water and sediment samples.
Many PCR-based tests are developed based on nuclear ribosomal
and mitochondrial gene
sequences (including tests from this study). Genes evolve at
different rates and a suitable DNA
region should vary in sequence sufficiently to allow the
identification of an individual to the
taxonomic level required. For specific identification, the DNA
marker should exhibit little or no
genetic variation within a species but differ sufficiently
between species so as to allow unequivocal
delineation.
In nuclear ribosomal genes and spacers, there is typically
little variation amongst individuals of a
species within and between populations (Larsen et al., 2005;
Livi et al., 2006). The ribosomal
DNA (rDNA) genes, Internal Transcribed Spacers (ITS) and
Intergenic Spacer (IGS)/ Non-
transcribed spacer (NTS) regions have been shown to be
particularly useful in defining species
specific markers for marine pest assay development. The
mitochondrial genome is also utilised for
diagnostic purposes; mitochondria are generally inherited
maternally making them particularly
useful as a species-specific marker for the delineation of
closely related species (e.g. Blair et al.,
2006, Kamikawa et al., 2008).
In this report we detail the development of qPCR assays for M.
senhousia and P. canaliculus, and
further specificity testing of the C. gibba qPCR assay developed
by Ophel-Keller et al. (2007).
7
-
Methods Quantitative PCR (qPCR) assay design Assays were
developed in the SARDI Diagnostics laboratory (see Figure 4) as
qPCR, using
TaqMan® minor groove binder (TaqMan MGB) chemistry. DNA
sequences of the desired genetic marker of target and related
organisms were imported into the sequence manipulation software
Bioedit (available from
http://www.mbio.ncsu.edu/RNaseP/info/programs/BIOEDIT/bioedit.html.),
and aligned using Clustal W. The genetic marker of choice is
defined by the ability for that marker
to delineate the target from heterologous species and also by
the availability of sequences from
publicly available databases. A range of DNA sequences was
obtained from the public domain
database GenBank (http://www.ncbi.nlm.nih.gov/genbank/). The
National Centre for Biotechnology
Information (NCBI), as a division of the National Library of
Medicine (NLM) at the National
Institutes of Health (NIH), has developed databases to deal with
molecular data, and facilitates the
use of molecular databases by the research and medical
community, Genbank is one of these
databases and is an annotated collection of all publicly
available nucleotide and amino acid
sequences.
Sequences of target and related taxa were aligned to infer
sequence regions that appeared to be
useful diagnostic regions. DNA sequences were identified which
were common to the target taxa
but where there were enough differences to distinguish target
from related taxa. Specific PCR
primers and TaqMan MGB probes were developed for target taxa
using the assay design software
Primer Express v2.0 (Applied Biosystems), an application that
designs primers and TaqMan MGB
probes that display suitable thermodynamic properties and
nucleotide content for efficient
amplification.
8
-
Figure 4: SARDI Diagnostics laboratory
Samples
Musculista senhousia samples (preserved in ethanol) were
obtained from Singapore (thanks to Dr
Maylene Loo) and M. senhousia DNA was kindly donated by Dr
Jawahar Patil (CSIRO). Perna
canaliculus samples were obtained frozen and purchased from an
Adelaide fish monger.Other
mytilid material was obtained through Dr Richard Willan,
Northern Territory Museum and Dr Paul
Fisher, Primary Industries and Fisheries, Queensland. Specimens
of Corbula gibba were
collected from Port Philip Bay for Ophel-Keller et al. (2007).
Specimens of Lentidium dalyfluvialis,
L. mediterraneum and Potamocorbula amurensis (Corbulidae)
preserved in RNAlater (Ambion),
were obtained from Mr Anders Hallan, University of Wollongong.
Other samples utilised for
specificity testing were collected whole (as part of other SARDI
marine pest qPCR projects), and
immediately stored frozen, or preserved in ethanol, for genomic
DNA (gDNA) extraction.
DNA extractions
gDNA was extracted from target and non-target samples by one of
two methods. The first method
was the Root Disease Testing Service (RDTS) commercial DNA
extraction method, a service
provided by SARDI Diagnostics, while the second method was the
QIAGEN DNeasy Blood and
9
-
10
Tissue kit following the manufacturer’s instructions. DNA
concentration was estimated by
fluorometry (Wallac 1420 multilabel counter) using Quant-iT™
PicoGreen® (Invitrogen). gDNA for
qPCR specificity experiments was typically diluted to 200
pg/μl.
Quantitative PCR qPCR reactions were carried out in 384 well
plates for analysis on an ABI HT 7900 sequence
detection system (Applied Biosystems, Foster City, CA) using
QuantiTect™ qPCR mastermix
(QIAGEN). Each qPCR assay was run with plate controls (no DNA
control and positive control for
each assay) and was analysed with ABI SDS 2.3 software (Applied
Biosystems). The PCR cycling
conditions were: 15 minutes at 95°C (activation) plus 40 cycles
of 15 secs at 95°C (denaturation)
and 60°C at 1 minute (annealing). qPCR results are given as
cycle threshold (Ct) values. The Ct
value represents the PCR cycle number at which the fluorescence
signal passes a fixed threshold,
displayed as a horizontal green line in plots showing number of
qPCR cycles vs magnitude of the
fluorescence signal intensity (∆Rn) (see Figures 5-7).
Results Primers and probes We designed a range of potential qPCR
assays for the detection and enumeration of M. senhousia
and P. canaliculus. Primers and TaqMan MGB probes that have
exhibited the highest level of
specificity to date are shown in Table 1. Other primer and probe
combinations that did not offer
appropriate specificity or amplification efficiency were not
considered further (data available on
request). Table 1 lists the genetic marker that the qPCR assay
targets, the nucleotide content of
the primers and probes, and the melting temperature (Tm) of the
primers and probes, which is
important for determining the reaction conditions of qPCR
experiments. The primers and probe for
the C. gibba qPCR assay (developed by Ophel-Keller et al., 2007)
are also shown in Table 1.
-
Tabl
e 1:
Prim
ers
and
TaqM
an M
GB
pro
bes
As
say
Gen
etic
M
arke
r Fo
rwar
d Pr
imer
(5’-3
’) Tm
(°
C)
Rev
erse
Prim
er (5
’-3’)
Tm
(°C
) Ta
qman
MG
B p
robe
Tm
(°
C)
Mus
culis
ta s
enho
usia
28
S
CG
GC
GG
TCA
GA
AG
CC
TGT
61
C
CA
GC
TATA
AA
CTC
CC
GA
CG
60
6FA
M- C
CG
GA
AG
GTG
AC
CC
G -M
GB
68
.1
Cor
bula
gib
ba
28S
G
GG
CA
GC
GTC
GTC
TTG
TG
61
CTA
TCG
GA
CTC
GTG
CC
TGTA
TTTA
G
58
6FA
M- A
TTC
CC
AA
AC
AA
CC
CG
- M
GB
68
Per
na c
anal
icul
us
IGS
C
GTA
ATC
CTC
AG
TAC
TGG
CTA
60
C
TCTA
AC
ATA
AG
GG
CTC
TAA
C
61
6FA
M- A
TAG
AG
TAG
AG
CTA
TTTA
GG
G -
MG
B
69.8
Key:
28S
-28S
ribo
som
al D
NA
. IG
S-In
terg
enic
spa
cer o
f mito
chon
dria
l DN
A. T
m-M
eltin
g te
mpe
ratu
re o
f prim
er/p
robe
. 6FA
M- 6
Car
boxy
fluor
esce
in (f
luor
opho
re),
MG
B-M
inor
Gro
ove
Bin
der n
on
fluor
esce
nt q
uenc
her.
11
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Musculista senhousia A TaqMan MGB PCR assay was designed for M.
senhousia by Ophel-Keller et al.,
(2007). Subsequent analysis of environmental samples collected
from Queensland
showed that the assay lacked specificity, so it has been
re-designed and re-
evaluated as part of this project. New primer and TaqMan MGB
probe combinations
were designed in silico, based on alignments of 28S rDNA
sequences, with the
intention of developing an assay specific to M. senhousia, based
on available
controls (see Table 1). The assay was screened against a wide
range of
heterologous controls, including mytilids, other bivalves and a
range of other
invertebrate species (see Table 2 and Figure 5). We note that
with one gDNA
sample of P. viridis, late amplification (i.e. ~ Ct of 40) was
exhibited (not shown).
However this result is not consistent across all our P. viridis
gDNA samples, and may
reflect a low level contamination of this sample with M.
senhousia gDNA.
12
-
Table 2: Results of specificity testing for M. senhousia qPCR
assay
Phylum Class Genus Species DNA (pg/ul) Ct values Mollusca
Bivalvia Musculista senhousia 200 19.90
Corbula gibba 200 UD Musculus cummingianus 200 UD Musculus
miranda 200 UD Perna viridis 200 UD Limnoperna securis 200 UD
Modiolus micropterus 200 UD Trichomya hirsutus 200 UD Perna
canaliculus 200 UD
Echinodermata Asterias amurensis 200 UD Chordata Ciona
intestinalis 200 UD
Ascidiella sp. 200 UD Arthropoda Crustacea Carcinus maenas 200
UD Annelida Polychaeta Sabella spallanzanii 200 UD
Undaria pinnatifida 200 UD NTC UD
\ Figure 5: Amplification plot for Musculista senhousia qPCR
assay specificity testing.
13
-
Corbula gibba A TaqMan MGB qPCR assay was designed for C. gibba
by Ophel-Keller et al.
(2007). While there have been no reported issues with the
specificity of the C. gibba
qPCR assay, it has not previously been adequately tested on DNA
from other
corbulid species
The C. gibba assay was further tested with the corbulid species:
Lentidium
dalyfluvialis, L. mediterraneum and Potamocorbula amurensis and
found not to
cross-react with any of these species (Table 3 and Figure 6).
Lack of specificity was
not encountered with the C. gibba qPCR assay during this
study.
Table 3: Results of specificity testing for C. gibba qPCR assay
Phylum Class Genus Species DNA (pg/ul) Ct values Mollusca Bivalvia
Corbula gibba 200 20.73
Lentidium dalyfluvialis 200 UD L. mediterraneum 200 UD
Potamocorbula amurensis 200 UD Musculista senhousia 200 UD Musculus
cummingianus 200 UD Musculus miranda 200 UD Perna viridis 200 UD
Limnoperna securis 200 UD Modiolus micropterus 200 UD Trichomya
hirsutus 200 UD Perna canaliculus 200 UD
Echinodermata Asterias amurensis 200 UD Chordata Ciona
intestinalis 200 UD
Ascidiella sp. 200 UD Arthropoda Crustacea Carcinus maenas 200
UD
Sabella spallanzanii 200 UD Undaria pinnatifida 200 UD NTC
UD
14
-
Figure 6: Amplification plot for Corbula gibba qPCR assay
specificity testing.
15
-
Perna canaliculus Blair et al. (2006) designed an end-point PCR
assay for the specific detection of
Perna canaliculus based on mitochondrial gene sequences. The
assay was modified
from Blair et al. (2006), utilising their specific reverse
primer and designing a new
forward primer and Taqman MGB probe suitable for use as a qPCR
assay. We have
tested the P. canaliculus qPCR assay with a wide range of
heterologous taxa,
including Perna viridis. We attempted to obtain sample of P.
perna from South Africa
but were unsuccessful. The P. canaliculus qPCR assay did not
cross react with the
range of invertebrate taxa shown in Table 4 and Figure 7.
Table 4: Results of specificity testing for P. canaliculus qPCR
assay
Phylum Class Genus Species DNA (pg/ul) Ct values Mollusca
Bivalvia Perna canaliculus 200 22.37
Corbula gibba 200 UD Musculus cummingianus 200 UD Musculus
miranda 200 UD Perna viridis 200 UD Limnoperna securis 200 UD
Modiolus micropterus 200 UD Trichomya hirsutus 200 UD Musculista
senhousia 200 UD
Echinodermata Asterias amurensis 200 UD Chordata Ciona
intestinalis 200 UD
Ascidiella sp. 200 UD Arthropoda Crustacea Carcinus maenas 200
UD
Sabella spallanzanii 200 UD Undaria pinnatifida 200 UD NTC
UD
16
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Figure 7: Amplification plot for Perna canaliculus qPCR assay
specificity testing
Discussion Quantitative PCR (qPCR) assays have been designed for
M. senhousia, C. gibba
and P. canaliculus. These assays have been designed in TaqMan
MGB format and
have been tested against a range of target and heterologous
taxa.
Ophel-Keller et al. (2007) developed a TaqMan MGB qPCR assay for
M. senhousia,
but subsequent testing of plankton samples from Queensland
indicated that there
were specificity issues with it. This study set out to design a
new M. senhousia
qPCR assay; once we had designed a putative assay the first step
was sourcing new
target material from Singapore and gDNA from CSIRO Marine and
Atmospheric
Research. Closely related native mytilids were also sourced for
specificity testing.
Based on available specimens it appears that the qPCR assay
described here is
specific to M. senhousia gDNA.
17
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We adapted an end-point PCR assay for P. canaliculus (see Blair
et al., 2006) to a
qPCR assay suitable for routine use in the SARDI Diagnostics
laboratory. The P.
canaliculus qPCR assay is specific, based on available
heterologous specimens,
including a range of mytilids. Testing the P. canaliculus qPCR
assay with Perna
perna would be advantageous and efforts to source material will
continue. It should
be noted that the end-point PCR developed by Blair et al. (2006)
was specific to P.
canaliculus and we have utilised the specific element (reverse
primer) of that assay
nd developed the forward primer and TaqMan MGB probe to be
specific to P.
m Anders
allan, University of Wollongong and the C. gibba qPCR assay did
not cross react
ith any of these specimens, or a range of other heterologous
specimens.
ip with Biosecurity SA at SARDI’s Diagnostic laboratories; qPCR
assays
eveloped for marine pests (including for this study) will be
utilised for routine use at
. The
. senhousia, P. canaliculus and C. gibba qPCR assays will be
utilised for the
a
canaliculus.
The C. gibba qPCR assay was developed by Ophel-Keller et al.
(2007). While this
assay did not exhibit any specificity issues when first
developed, there was a need to
test the assay with other corbulid species. Specimens were
obtained fro
H
w
Conclusions and future approaches With continued development of
these qPCR assays, in conjunction with the
development of assays for other significant marine pest species,
it is feasible that
comprehensive surveillance for marine pests in South Australia
can be achieved
using DNA based assays. Molecular-based testing of environmental
samples (water
and sediments) offers the potential for more rapid and cost
effective testing than
more resource and time intensive traditional sampling methods.
The Australian
Testing Centre for Marine Pests (ATCMP) is proposed for
establishment in
partnersh
d
ATCMP.
In a parallel project (also funded by Biosecurity SA), SARDI has
been developing a
plankton collection method, in which samples are filtered and
preserved for later
molecular analyses. It is anticipated that this method can be
used, in conjunction
with qPCR, to detect a broad range of pest species for
surveillance purposes
M
detection of DNA of target larval stages using this plankton
collection strategy.
18
-
Specific diagnosis of marine pests is central to: (a) rapidly
establishing the
prevalence and distribution of marine pest species in the
environment in conjunction
with traditional sampling and taxonomic techniques; (b)
monitoring changes in marine
pest distribution spatially and temporally; and (c) conducting
targeted eradication and
control programmes if economics and logistics permit.
19
-
References Bax N., Williamson A., Aguero M., Gonzalez E. and
Geeves W. (2003) Marine invasive alien species: a threat to global
biodiversity. Marine Polution 27: 313-23. Blair D., Waycott M.,
Byrne L., Dunshea G., Smith-Keune C., Neil KM. (2006) Molecular
discrimination of Perna (Mollusca: Bivalvia) species using the
polymerase chain reaction and species-specific mitochondrial
primers. Marine Biotechnology. 8: 380-385. Bott N.J., Ophel-Keller
K.M,. Sierp M.T., Herdina, Rowling K.P., McKay A.C., Loo M.G.K.,
Tanner J.E. and Deveney M.R. (2010). Toward routine, DNA-based
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Marine Pollution Bulletin 38, 36-43. Dexter, D.M., and Crooks, J.A.
(2000) Subtidal benthic communities and the invasion of an exotic
mussel in an urbanized southern California bay: A long-term
history. Bulletin Southern California Academy of Science 99,
128-146. Espy M.J., Uhl J.R., Sloan L.M., Buckwater S.P., Jones
M.F. et al. (2006) Real-time PCR in clinical microbiology:
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aliens and biodiversity in the Mediterranean Sea. Marine Pollution
Bulletin 55: 314–322. Galluzzi L, Penna A, Bertozzini E, Vila M,
Garcés E and Magnani M. (2004) Development of a real-time PCR assay
for rapid detection and quantification of Alexandrium minutum (a
dinoflagellate). Applied and Environmental Microbiology 70:
1199-206. Hayes K. R. and Sliwa C. (2003) Identifying potential
marine pests- a deductive approach applied to Australia. Marine
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and Sako Y. (2008) Development of a novel molecular marker on the
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Livi S., Cordisco C., Damiani C., Romanelli M. and Crosetti D.
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McKay A. (2007) Development of gene probes for introduced marine
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Slack-Smith, S.M., A. Brearley. (1987) Musculista senhousia
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21
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22
Glossary of Terms ATCMP- Australian Testing Centre for Marine
Pests. CCIMPE- Consultative Committee on Introduced Marine Pest
Emergencies. Ct-Cycle threshold: qPCR cycle where fluorescence is
observed above a threshold level indicating a positive result. DNA-
Deoxyribonucleic Acid: genetic information responsible for the
development and function of all organisms, with the exception of
some viruses. gDNA-genomic Deoxyribonucleic Acid: the total DNA of
an organism, or the genome of an organism. IGS- Intergenic Spacer
of mitochondrial DNA. ITS-2: second Internal Transcribed Spacer: a
region of ribosomal DNA that does not code for any genes. mtDNA-
Mitochondrial DNA: the genome of the intracellular organelles
called mitochondria. Considered an informative diagnostic region.
NIMPCG- National Introduced Marine Pest Coordinating Group NTC- No
Template Control: a PCR reaction with no DNA template added, is
used to ensure that PCR is not previously contaminated i.e. NTC
should not be a positive result. Nucleotide: Molecules, that when
joined together make up the functional units of DNA. PCR-
Polymerase Chain Reaction: enzymatic technique used for the
amplification of nucleic acids (e.g. DNA). qPCR- Quantitative
Polymerase Chain Reaction: PCR reaction whereby amplification is
monitored in real time through the use of fluorescent dyes or probe
based chemistry. TaqMan MGB probe-TaqMan Minor Groove Binder probe:
hybridises to specific fragment of DNA and emits fluorescence; used
to quantify target DNA in a sample. rDNA- ribosomal
Deoxyribonucleic Acid: codes for vital cellular components in
Eukaryotes; an informative diagnostic marker. RDTS- Root Disease
Testing Service: a commercial diagnostic service at SARDI.
Executive SummaryAcknowledgementsIntroductionMusculista
senhousiaCorbula gibbaPerna canaliculusMolecular testing methods
for marine pests
MethodsQuantitative PCR (qPCR) assay designSamplesDNA
extractionsQuantitative PCR
ResultsPrimers and probesMusculista senhousiaCorbula gibbaPerna
canaliculus
DiscussionConclusions and future approaches
ReferencesGlossary of Terms
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